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HER2-positive breast cancer accounts for 25% of all cases and has a poor prognosis. Although progress has been made in understanding signal transduction, little is known of how HER2 achieves gene regulation. We performed whole genome expression analysis on a HER2+ and HER2− breast cancer cell lines and compared these results to expression in 812 primary tumors stratified by their HER2 expression level. Chip-on-chip with anti-RNA polymerase II was compared among breast cancer cell lines to identify genes that are potentially activated by HER2. The expression levels of these HER2-dependent POL II binding genes were determined for the 812 HER2+/− breast cancer tissues. Genes differentially expressed between HER2+/− cell lines were generally regulated in the same direction as in breast cancer tissues. We identified genes that had POLII binding in HER2+ cell lines, but without significant gene expression. Of 737 such genes “poised” for expression in cell lines, 113 genes were significantly differentially expressed in breast tumors in a HER2-dependent manner. Pathway analysis of these 113 genes revealed that a large group of genes were associated with stem cell and progenitor cell control as indicated by networks centered on NANOG, SOX2, OCT3/4. HER2 directs POL II binding to a large number of genes in breast cancer cells. A “poised” class of genes in HER2+ cell lines with POLII binding and low RNA expression but is differentially expressed in primary tumors, strongly suggests a role of the microenvironment and further suggests a role for stem cells proliferation in HER2-regulated breast cancer tissue.

We optically measured effects of orally available ivacaftor (Kalydeco®) on sweat rates of identified glands in 3 R117H subjects, each having a unique set of additional mutations, and compared them with 5 healthy control subjects tested contemporaneously. We injected β-adrenergic agonists intradermally to stimulate CFTR-dependent 'C-sweat' and methacholine to stimulate 'M-sweat', which persists in CF subjects. We focused on an R117H-7T/F508del subject who produced quantifiable C-sweat off ivacaftor and was available for 1 blinded, 3 off ivacaftor, and 3 on ivacaftor tests, allowing us to estimate in vivo fold-increase in sweat rates produced by ivacaftor's effect on the open probability (PO) of R117H-CFTR. Measured sweat rates must be corrected for sweat losses. With estimated sweat losses of 0.023 to 0.08 nl·gland-1·min-1, ivacaftor increased the average C-sweat rates 3-7 fold, and estimated function as % of WT were 4.1-12% off ivacaftor and 21.9-32% on ivacaftor (larger values reflect increased loss estimates). Based on single tests, an R117H-7T/ R117H-7T subject showed 6-9% WT function off ivacaftor and 28-43% on ivacaftor. Repeat testing of an R117H-5T/F508del subject detected only trace responding to ivacaftor. We conclude that in vivo, R117H PO is strongly increased by ivacaftor, but channel number, mainly determined by variable deletion of exon 10, has a marked influence on outcomes.

To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

Background. Herceptin, which targets the protein HER2, has poor efficacy as a monotherapy. To better understand the role of HER2, we compared isogenic HER2 expressing and non-expressing cells for the distribution for RNA polymerase II (POL II) on promoters and RNA expression. Methods. Chromatin immunoprecipitation was performed with anti-RNA polymerase II (POL II) in high HER2-expressing and non-expressing MCF7 cells as well as two breast cancer cell lines with naturally amplified HER2; MDA453 and BT474. Binding of POL II was assessed on Agilent promoter arrays. Gene activation in the same cells was assessed by expression analysis using U133plus2 arrays. Results. De novo expression of HER2 in MCF7 cells causes POLII to interact strongly with 606 genes not bound in empty vector control cells (p < 0.05). 30% of these genes exhibited a significant increase in transcription (p < 0.05; Fold-change > 1.4). POL II also interacts 1079 genes that are common between HER2-expressing and control cells. Conversely, 678 genes were bound more tightly by POL II in the control (p < 0.05). 20% of these genes exhibited a significant decrease in transcription. An additional 825 genes, bind POLII “loosely” in MCF7HER2 cells but not in MCF7 cells. 30% of these genes were significantly differentially regulated relative to control. The transcription of 68 loosely bound genes was tested by qPCR for all three cell lines. When compared with the array data, the correlation coefficients were 0.74 - 0.90;(p < 0.01). In both HER2 and non-expressing cells, loosely bound POL II sites are largely associated with 3′ positions of the Agilent probes within the transcribed region, whereas strong binding sites were largely localized to + 250 bp of the transcription start sites (TSSs). The transcriptome of MCF7 was compared to MCF7HER2 and the two HER2-expressing cancer cell lines. 633 genes were differentially expressed between the control and the three lines. Conclusions. The expression of HER2 is associated with a marked alteration in the number of genes that interact with POL II, involving hundreds of genes. The resulting gene activity depends upon the binding strength of POL II. Strong localized binding in one part of the promoter is often associated with less transcription, and diffused looser binding is often associated with higher transcription, whereas a lack of POL II binding is usually associated with no transcription. This is consistent with a mass action model in which transcription is maximal for POL II sites with an optimal koff rate and declines with either an increase (stalled) or decrease (not interacting) in binding. 42%, of the HER2-dependent gene interactions and transcription changes occur in both of the human cancer cell lines with naturally amplified HER2. These observations reveal a much expanded role of HER2 in gene regulation and indicate a greatly extended role in gene activation in HER2-positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3001. doi:1538-7445.AM2012-3001